Ivaldo I. Junior

A Comprehensive Study on the Activity and Deactivation of Immobilized Lecitase Ultra in Esterifications of Food Waste Streams to Monoacylglycerols

Lecitase Ultra was immobilized on Amberlites XAD2 and XAD4, through physical entrapping under conventional stirring or ultrasound irradiation, and characterized by standard techniques. The resulting immobilized biocatalysts were utilized in the valorization of an acidic food-derived residue from a palm oil refining process to produce monoacylglycerols from isopropylidene glycerol under batch and continuous flow conditions. Results indicated that the immobilized biocatalysts could moderately convert the food waste residue (max. conversion 50-60 %), exhibiting interesting stability under continuous flow conditions.

Ceanothane and Lupane Type Triterpenes from Zizyphus joazeiro– An Anti-Staphylococcal Evaluation

The present paper describes the phytochemical and anti-staphylococcal activity investigation of the dichloromethane extract of the Brazilian plant Zizyphus joazeiro Mart. The purification steps were guided by bioassays against 17 bacterial strains of clinical sources, including methicillin-resistant (MRSA) and -sensitive (MSSA) Staphylococcus aureus as well as MRSA (ATCC 33591) and MSSA (ATCC 29213) reference strains. One of the more active fractions is comprised of three lupane-type triterpenes, the methylbetulinate (1) as well as the known betulinic (2) and alphitolic (3) acids and, for the first time in the Z. joazeiro, two ceanothane type triterpenes, the methylceanothate (4) and the epigouanic acid A (5). These substances were assayed against one clinical (PVL+) and the reference strains of S. aureus as well as the ATTC 12228 strain of S. epidermidis, in concentrations that varied from 128 to 0.125 microg/mL in order to establish the minimum inhibitory concentration (MIC) of the drugs. The minimum bactericide concentration (MBC) was also evaluated to distinguish the bactericidal from bacteriostatic activity of the crude fractions and single compounds. Compounds 3 and 4 possess the highest antibacterial activity. They inhibit all bacteria tested at 32 microg/mL and 16 microg/mL, respectively, while the other compounds showed no activity at 128 microg/mL. In contrast to single compounds, the triterpenoid fraction showed bactericidal activity at 256 microg/mL. Structural elucidations are based on 1D and 2D NMR spectroscopy as well as HR-FT-ICR-MS experiments.

Sporopollenin as an efficient green support for covalent immobilization of a lipase

Sporopollenin exine capsules (SECs), derived from the spores of Lycopodium clavatum, have been functionalised with 1,n-diamines and the resulting aminoalkyl microcapsules used to immobilize Candida antarctica lipase B (Cal B) via a glutaradehyde-based diimine covalent linker. The supported enzyme efficiently catalyzes the esterification of oleic acid with ethanol. Initial rates using the SEC-CalBs were comparable to the commercial enzyme Novozym 435, but displayed up to 20-fold higher specific activity. The supported enzymes could also be recycled and after four cycles displayed only a modest decrease in conversions. In a kinetic resolution the SEC-CalBs efficiently acetylated rac-1-phenylethanol, with conversions up to 37% after 5 hours and product enantiomeric excesses of >99%. Related to this, the dynamic resolution of rac-1-phenylethylamine, in the presence of Pd–BaSO4 and ammonium formate, led to the acetylated amine with a 94% conversion and >99% ee.

Consecutive lipase immobilization and glycerol carbonate production under continuous-flow conditions

Several value-added products can be produced from glycerol and among these products glycerol carbonate (GC) has received much attention in recent years because of its physical properties and wide application in the chemical, pharmaceutical and food industries. As a continuation of our efforts to produce GC under environmentally acceptable conditions, we proposed a consecutive approach where lipase B from Candida antarctica was immobilized on Accurel MP1000 (CalBAcc) under continuous-flow conditions followed by glycerol carbonate production by a cascade process of triacylglycerol hydrolysis, biodiesel synthesis and esterification of the remaining glycerol with dimethyl carbonate towards GC production. Results of the immobilization of lipase B from Candida antarctica on a continuous-flow protocol demonstrated that a short residence time of 25 minutes could lead to 83% of protein loading. Screening of glycerol carbonate production in a packed bed reactor revealed that CalBAcc and N435 showed the best results of conversion and selectivity, with more selectivity by CalBAcc, that could lead to quantitative yields and excellent selectivities of the desired glycerol carbonate at residence times of 176 minutes when working with soybean and palm oil. Comparatively, the results obtained with CalBAcc were better than the ones with Novozym 435 since no full conversion was observed.

Lipase immobilization towards improved productivity on kinetic resolutions by a continuous-flow process

Development of asymmetric transformations for the synthesis of chiral molecules has achieved great results in the last decade. Besides the great evolution achieved during recent years in the biotechnology and biocatalysis fields, industrial processes using enzymatic approaches are few and a more broad application of such technology towards the synthesis of chiral molecules is still under development. Herein we report our results on the immobilization of lipase B from Candida antarctica on five different commercial supports for the development of a continuous-flow kinetic resolution of alcohol. Immobilization on Accurel MP1000 gave the best results arriving at productivities of around 140 g per h per g of protein and the immobilized biocatalyst could be recycled 5 times without significant loss of activity.

Novel nanoparticle/enzyme biosilicified nanohybrids for advanced heterogeneously catalyzed protocols

Novel bio-nanohybrids based on room temperature one-pot synthesized lipase-nanoparticle systems were developed and characterized in this work, with subsequent investigations of their catalytic activities and stability as compared to the free enzymes. Preliminary results revealed excellent stabilities, solvent tolerance, and activities as compared to free lipases, opening up further scenarios for their utilization under continuous flow conditions as well as in tandem reactions.

Cellulose as an Efficient Matrix for Lipase and Transaminase Immobilization

Immobilization of enzymes is important to improve their stability and to facilitate their recyclability, aiming to make biocatalytic processes more efficient. One of the important aspects is the utilization of cheap, abundant, and environmentally friendly carriers for enzyme immobilization. Here we report the use of functionalized cellulose for lipase and transaminase immobilization. High immobilization efficiencies (up to 90%) could be achieved for the transaminase from Vibrio fluvialis. For immobilized lipase CAL-B as well as the transaminase, good conversions and recyclability could be demonstrated in kinetic resolutions to afford chiral alcohols or amines. Moreover, such application of the immobilized transaminase enabled very high conversions in a continuous-flow process in the asymmetric synthesis of (S)-phenylethylamine (80% conversion, >99% ee).

Nanoencapsulated Lecitase Ultra and Thermomyces lanuginosus Lipase, a Comparative Structural Study.

Two commercially available and widely used enzymes, the parent Thermomyces lanuginosus lipase (TLL) and the shuffled phospholipase A1 Lecitase (Lecitase Ultra), were encapsulated in AOT/isooctane reverse micelles and evaluated regarding their structure and activity. Preparations were also tested as effective biocatalysts. Small-angle X-ray scattering (SAXS), electronic paramagnetic resonance (EPR), and fluorescence spectroscopy were the techniques applied to assess the effects of enzyme incorporation to a reverse micellar nanostructure. SAXS analysis showed that the radius of gyration (Rg) changed from 16 to 38 Å, as the water content (w0) increased. Elongated shapes were more commonly observed than spherical shapes after enzyme encapsulation. EPR studies indicated that enzymes do not participate in the interface, being located in the aqueous center. Fluorescence energy transfer showed that TLL is located in the water core, whereas Lecitase Ultra is closer to the interface. Enzymatic activity toward a standard esterification reaction endured after the enzyme was incorporated into the micelles. The activity of TLL for systems with w0 15 showed the highest conversion yield, 38% in 2 h, while the system with w0 10 showed the highest initial velocity, 0.43 μM/min. This last system had a Rg of 19.3 Å, similar to that of the TLL monomer. Lecitase Ultra showed the highest conversion yields in systems with w0 10, 55% in 2 h. However, the initial rate was much lower than that of TLL, suggesting less affinity for the substrates, which is expected since Lecitase Ultra is a phospholipase. In summary, we here used several spectroscopic and scattering techniques to reveal the shape and stability of TTL and Lecitase Ultra encapsulated systems, which allowed the selection of w0 values to provide optimized enzymatic activity.

Improving the esterification activity of Pseudomonas fluorescens and Burkholderia cepacia lipases via cross-linked cyclodextrin immobilization

The search for a new, efficient and sustainable matrix for biocatalyst immobilization is a growing area in biotechnology. Our proposed approach deals with the utilization of solid cross-linked β-cyclodextrin as supports for enzyme immobilization. Results obtained in terms of enzyme activity and thermal stability of novel immobilised materials have been found to remarkably improve those obtained using commercial immobilized enzymes in esterification reactions (e.g., monostearin synthesis).

Pentaclethra macroloba tannins fractions active against methicillin-resistant staphylococcal and Gram-negative strains showing selective toxicity

The ethanol extract of the vegetal species Pentaclethra macroloba (Willd.) Kuntze, Fabaceae, was fractioned and the antibacterial activity was determined. The active ethyl acetate (ea) fraction showed activity against Gram-positive (Staphylococcus spp. and Enterococcus spp.) and Gram-negative (Pseudomonas aeruginosa, Acinetobacter spp. and Klebsiella pneumoniae) multiresistant bacteria. Gallic acid derivatives were identified as the main compounds in inactive subfractions from the ea fraction, while the active one afforded ellagic acid as the major constituent when submitted to acid hydrolysis reaction, which suggests the presence of hydrolysable tannins. The minimum bactericidal concentration analysis showed a bactericide mechanism of action for the tannin subfraction found. The antibacterial mechanism of action of the active tannin subfraction against S. aureus reference strains (ATCC 29213 e 33591) was proposed adopting an in vitro assay of protein synthesis inhibition. For this, bacterial cells were labeled with [35S] methionine in the presence of the subfraction. The protein synthesis inhibition was observed at 256 µg/mL of this subfraction. At this concentration it did not present cytotoxicity in eukaryotic cells by the neutral red technique, suggesting selective toxicity. The present study is the first in vitro investigation of the antibacterial properties of tannin fractions obtained from a polar extract of P. macroloba.